Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool

Graphical Abstract A broad host-range CRISPR-Cas9 delivery tool was developed and used to conjugatively remove antimicrobial resistance (AMR) plasmids (top) and protect a series of coliform and Pseudomonas isolates from AMR plasmid uptake (bottom).


INTRODUCTION
Antimicrobial resistance (AMR) is one of the key challenges facing modern-day healthcare: at least 1.2 million deaths were directly attributed to bacterial AMR worldwide in 2019 [1]. Selection for AMR genes can occur at low concentrations of antibiotics in the environment [2], as well as during chemotherapy in humans and animals. To compound this issue, many AMR genes are easily transferred between different bacterial taxa by horizontal gene transfer, predominantly by plasmid transfer (reviewed in [3]). In this way, previously susceptible pathogens may gain resistance to antibiotics.
Blocking plasmid uptake in key pathogens and environmental bacteria, or removing resident plasmids from these, may provide a means of preventing or decreasing the level of antimicrobial-resistant bacterial infections (reviewed in [4]). One approach of reversing resistance in target bacteria through AMR plasmid removal is the use of CRISPR-Cas9 or related minimal CRISPR systems, typically delivered on an engineered plasmid by conjugation or by means of an engineered bacteriophage. After uptake of the CRISPR delivery tool by recipient cells, the Cas9 nuclease cleaves a DNA sequence defined by its single guide RNA (sgRNA). Depending on the target gene location, this leads to chromosome cleavage and cell death, or plasmid cleavage and resensitization to antibiotics (reviewed in [5,6]).
CRISPR delivery tools have been engineered using various genetic backbones, for instance non-replicating phage plasmids (phagemids) [7,8], expression vectors [9], or synthetic conjugative or mobilizable plasmids [10,11]. These are effective at blocking transfer of resistance genes into specific strains and can remove AMR genes from them. However, in nature, bacteria are commonly embedded in complex microbial communities consisting of many different bacterial strains and species, in which AMR plasmids can be found in multiple, sometimes phylogenetically distant, strains (epidemic plasmids [12]). To target AMR plasmids across different strains or even species, the currently available narrow host-range CRISPR-Cas delivery tools would need to be individually engineered for each target strain. To overcome such drawbacks, a broad host-range delivery vehicle that can be naturally transferred to a range of bacterial species would be highly suitable for application in bacterial communities.
To address this issue, we sought to design a mobile, broad host-range CRISPR-Cas9 expression system that can block AMR gene uptake in multiple species.
We chose the IncP-1ε plasmid pKJK5 [13] as a template for our CRISPR delivery tool. pKJK5 was previously shown to have a particularly broad host range and to spread effectively through microbial communities derived from soil, pig gut microbiomes and wastewater treatment plants. Using either Escherichia coli, Pseudomonas putida, or Kluyvera sp. as donor species, the plasmid was taken up by species belonging to at least 11 different phyla of both Gram-negative and Gram-positive bacteria [14][15][16]. The gentamicin resistance-encoding cloning vector pHERD30T [17] was chosen as a target plasmid for proof-of-concept experiments, as it can be maintained by Escherichia and Pseudomonas spp., is compatible with pKJK5, and encodes no stability genes that may interfere with CRISPR-Cas9 targeting.
Here, we engineer pKJK5 to encode cas9 and sgRNA. We show that this engineered CRISPR-Cas9 delivery tool can be used to protect target cells from AMR plasmid uptake, to remove resident AMR plasmids and apply this tool in a range of bacterial species and isolates.

RESULTS
We engineered the broad host-range plasmid pKJK5 to carry a CRISPR-Cas9 cassette programmed to block uptake of pHERD30T, a plasmid-encoding Gentamicin resistance gene aacC1.

pKJK5::csg construction
First, we designed a CRISPR-Cas9 entry cassette in silico that can be recombined with pKJK5. The gene cassette was designed to include the nuclease-encoding gene cas9, sgRNA, which determines its targeting specificity, and GFP (green fluorescent protein) to track plasmid transfer (Fig. 1a). Strategic restriction sites were incorporated in the gene cassette design to ensure full modularity Gene lengths are to scale; spacings, restriction sites, promoters, terminators and ribosome binding sites are not. (c) sgRNA region in detail. Highlighted in red: nucleotide mutations introduced in upper stem region to form SacI restriction site. The region to be exchanged for N20 specificity exchange is indicated with blue crossover lines. (d) Homologous recombineering allowed insertion of the CRISPR-Cas9 cassette into dfrA, disrupting this gene in pKJK5's accessory gene load. See the Methods section for details. (Fig. 1b) . The sgRNA gene was edited to allow simple exchange of the specificity-defining 20 nt stretch (N20) (Fig. 1c). GFP was added under control of the lacI-repressible promoter P A1/04/03 [18] to allow optional repression of GFP expression. The entire CRISPR-Cas9 entry cassette was flanked by homology arms matching the trimethoprim resistance gene dfrA on pKJK5 to allow homologous recombination.
This gene cassette was recombined with pKJK5 using homologous λ-red recombineering to yield pKJK5::csg (encoding the genes cas9, sgRNA, GFP; see (Fig. 1d) and Methods section). To test the ability of pKJK5::csg to target AMR plasmids, we generated two pKJK5::csg variants with different sgRNA specificities: pKJK5::csg[aacC1] targets gentamicin resistance gene aacC1 on pHERD30T. As a non-targeting (nt) control, pKJK5::csg[nt] carries a sgRNA with a random nucleotide sequence not present in the study system.
The nucleotide sequence of pKJK5::csg[aacC1], determined by Illumina sequencing, is published on GenBank under accession number OP921802.

pKJK5::csg acts as a barrier to AMR plasmid acquisition
To test whether pKJK5::csg can act as a barrier to plasmid acquisition, we measured the transformation efficiency of a targeted plasmid (pHERD30T) or an untargeted control plasmid (pHERD20T) in E. coli carrying pKJK5::csg[aacC1]/[nt]. Instead of pHERD30T's aacC1 gentamicin resistance gene, pHERD20T encodes ampicillin resistance gene blaTEM and is not targeted by either sgRNA. Accordingly, the control plasmid's transformation efficiency was high regardless of pKJK5's sgRNA specificity (~10 6 c.f.u. ml −1 µg −1 DNA; Fig. 2). In contrast, for the targeted plasmid no successful transformants of DH5α+pKJK5::csg[aacC1] could be recovered. The same plasmid showed transformation efficiencies of ~10 4 c.f.u. ml −1 µg −1 DNA in DH5α+pKJK5::csg[nt]. This means that transformation efficiency of a targeted plasmid was reduced to at least the limit of detection (4 c.f.u. ml −1 µg −1 [aacC1] transformation with pHERD30T did not yield any transformants, data points are displayed as ½ of the limit of detection. Grey box, data points underneath the limit of detection. n, 6, diamonds, mean, circles, individual data points. DNA) in the presence of a targeting CRISPR-Cas system and was nearly four orders of magnitude lower than in the presence of the non-targeting control.

pKJK5::csg can conjugatively remove resident plasmids
Having established that pKJK5::csg is able to prevent AMR plasmid uptake, we then tested whether it also allowed removal of resident AMR plasmids from target bacterial strains.
To test this, we allowed both versions of pKJK5::csg (targeting and non-targeting) to transfer from an E. coli DH5ɑ donor to target bacterium E. coli K12 carrying plasmid pHERD30T by mixing overnight recipient and donor cultures in liquid matings.
Raw colony counts revealed that while overall cell densities ('LB') were slightly lower for the non-targeting control, the number of recipients ('K') was significantly lower when pKJK5::csg[aacC1] was delivered compared with its non-targeting counterpart ( Fig. 3b; P<0.001; F=273.9, df=11 and 60, R 2 =0.98). This reveals a possible fitness cost of CRISPR-mediated AMR plasmid removal.
Overall, the conjugative delivery of pKJK5::csg using a donor strain led to CRISPR-mediated removal of a targeted plasmid from part of a recipient population.

pKJK5::csg is a broad host-range barrier to plasmid acquisition
Finally, we tested the ability of pKJK5::csg to act as a barrier to plasmid acquisition in a broader range of bacterial species. To this end, pKJK5::csg carrying transconjugants originating from several environmental, animal and human-associated coliform isolates, as well as of two species of Pseudomonas (Table S1), were each transformed with pHERD30T.
Transformation of those isolates carrying pKJK5::csg[nt] was successful (~666-4320 c.f.u. ml −1 µg −1 DNA; Fig. 4). In contrast, transformation efficiency of all isolates carrying pKJK5::csg[aacC1] was below, or in few individual replicates immediately above the limits of detection. Therefore, transformation efficiency of pHERD30T when carrying pKJK5::csg[aacC1] remained at least two-three orders of magnitude below the transformation efficiency recorded when carrying pKJK5::csg[nt] in all isolates (P<0.001; F=347.6; df=11 and 48; adjusted R 2 =0.99). Additionally, we tested whether pKJK5::csg can be conjugatively delivered to these strains to remove pHERD30T as a resident plasmid. After delivery of pKJK5::csg by liquid mating or filter mating (which is predicted to increase conjugation efficiency [19]) using E. coli donors, most isolates did not show a drop in target plasmid maintenance (Fig. S1, available in the online version of this article). The exception to this was isolate TV1-2, in which we observed modest pHERD30T removal after filter mating ( Fig. S2; 63.9±32.8 % after non-targeting vs 24.8±19.3 % after targeting treatment; P=0.016 as assessed by a binomial GLM and Tukey's post-hoc analysis). Despite undetectable plasmid removal at a population level, a drop of target plasmid maintenance in all coliform isolates was seen when specifically assessing the transconjugant proportion of the population ( Fig. S3; P<0.05 for all isolates except those with transconjugant formation close to or below the limit of detection; assessed by Gaussian GLM and Tukey's post-hoc test).
Overall, pKJK5::csg proved to be an efficient barrier to uptake of a plasmid containing a targeted AMR gene. This was effective in a range of species of laboratory as well as environmental, animal-, and human-associated isolates without the need for re-engineering of pKJK5::csg. While conjugative removal of a resident plasmid from these isolates was undetectable or modest on a population level, target plasmid removal in transconjugants confirmed pKJK5::csg activity after conjugation in this isolate library.

DISCUSSION
Several previous studies aimed to resensitize bacteria by conjugatively delivering an engineered CRISPR plasmid [10,11,[20][21][22][23], but most of these deployed a mobilizable CRISPR delivery tool that requires either a second conjugative plasmid or an engineered donor strain [11,20,[22][23][24]. Crucially, the application of a CRISPR delivery tool has been shown to be more effective when conjugative machinery and CRISPR machinery are encoded on the same genetic element (in cis), rather than on separate plasmids (in trans) [25]. Therefore, we generated the fully conjugative plasmid pKJK5::csg, and demonstrated that it protects a range of host species from uptake of targeted AMR plasmid pHERD30T. Additionally, conjugation of pKJK5::csg led to removal of pHERD30T from a recipient strain.
CRISPR delivery tools could provide a means of tackling hotspots of horizontal gene transfer and reservoirs of AMR genes by removal of AMR plasmids: the human gut microbiome [26] and environments such as livestock farms or wastewater [27] see frequent exchange of resistance genes between different bacterial species, including pathogens. Thanks to the broad natural host range of the plasmid it is derived from [14][15][16], pKJK5::csg is particularly promising for the application in such microbially diverse environments and could help to either remove AMR plasmids from them (as in Fig. 3), or to protect microbiomes from becoming colonized by AMR plasmids (as in Fig. 4). All genetic cargo of pKJK5::csg (cas9, sgRNA, GFP) was inserted into trimethoprim resistance gene dfrA, which sits within the accessory gene pool of pKJK5 [13]. Therefore, we can expect pKJK5's broad transfer range into at least 11 phyla to be maintained for pKJK5::csg.
In previous work, AMR plasmid removal was limited by the low conjugation efficiency of the CRISPR plasmid. In our work, conjugation efficiency was also modest: conjugation of pKJK5::csg to recipients failed in almost half of the cases (only ~61-65 % of recipients formed transconjugants; Fig. 3a). In contrast, once pKJK5::csg was present in target cells, CRISPR targeting only failed in ~1 in 5000 cases (~0.02 % of pKJK5::csg[aacC1] transconjugants also contained pHERD30T; Table S2). Furthermore, conjugation efficiency for the different isolates ( Fig. S4) varied depending on recipient identity. As pKJK5::csg was active in all cases once present in recipients (Fig. S3), we suggest that target plasmid removal using pKJK5::csg could primarily be improved by optimizing this CRISPR delivery tool's conjugation efficiency. For instance, identification of a suitable donor for a target community is paramount: conjugation of wild-type pKJK5 to a soil community was more effective using E. coli as a donor than when using Kluyvera sp. or Pseudomonas putida [14]. Furthermore, plasmids can evolve higher transfer rates as a trade-off against increased plasmid cost -as observed for R1 when cultured under conditions with ample naïve hosts [28]. Perhaps directed evolution could achieve the same for pKJK5::csg.
Although conjugation efficiency is likely to be a highly important factor for optimization, CRISPR-mediated target plasmid removal outcome may also depend on other variables. For example, in our work pKJK5::csg[aacC1] reduced the efficiency of target plasmid transformation compared with pKJK5::csg[nt] to different extents in different species (Fig. 4), which may be related to variation in pKJK5::csg fitness costs and maintenance. We previously observed this phenomenon for a closely related plasmid, pKJK5::gfp PL , the cost of which was associated with plasmid maintenance and varied between different hosts and growth contexts [29]. This plasmid cost is likely due to constitutive costs of Cas9 and sgRNA expression, which can have different extents in different species [30][31][32].
Beyond this, target plasmid removal efficiency by CRISPR delivery tools could depend on other factors, such as target plasmid mobility [11], plasmid copy number [33], or the presence of other payload genes on target plasmids or in target genomes such as anti-CRISPR proteins [34] or toxin-antitoxin systems. These should be further experimentally investigated for optimization of AMR plasmid removal using CRISPR-Cas9.
In summary, CRISPR delivery tools which target and cleave AMR plasmids may be used to protect their bacterial hosts from plasmid uptake or to resensitize them to antibiotics by removing resident plasmids. Proof-of-concept experiments have achieved this in simple set-ups, but in nature bacteria are embedded in complex communities so a broad host-range CRISPR delivery tool is needed. This work establishes pKJK5::csg as a broad host-range CRISPR delivery tool with the ability to remove AMR plasmids from diverse bacterial species, and thereby forms a basis for interventions aimed at clearing AMR genes from bacterial communities.
Where E. coli MFDpir was used, cultures were supplemented with 300 mM DAP (diaminopimelic acid) to ensure growth of this auxotrophic strain. By omitting DAP, the strain could be selected against.
Pig faeces isolate bhiF2 was isolated from a microbial pig gut community. Briefly, pig faecal samples, collected from four Cornish black pigs, were suspended in 10 % glycerol and 0.9 % (w/v) NaCl, and subsequently blended and strained. The resulting pig faeces slurry was plated onto BHI (brain heart infusion) agar plates without selection, and bhiF2 was one of several visually distinct bacterial isolates picked from these plates. Genus identity was confirmed as Escherichia/Shigella by 16S colony PCR, Sanger sequencing and blast homology search.
All molecular cloning steps were carried out with high-fidelity restriction enzymes (NEB) and according to manufacturer protocols, using commercially chemically competent E. coli DH5α cells (NEB).

In silico cassette construction and specificity swap
The CRISPR-Cas9 gene cassette was constructed and restriction sites were identified using Benchling [35]; an overview of the workflow is shown in Fig. 1. Sources of nucleotide sequences for each module are summarized in Table 2.
Genes were codon-optimized using OPTIMIZER [36] with pKJK5 codon usage database tables [37]. Common restriction sites were removed from these coding sequences by changing codons to the second most common on pKJK5. When creating or altering multiple cloning sites, random nucleotides were added to increase spacing and allow double digestions. Terminator presence (and absence from unwanted regions) was checked using Arnold [38].
The single guide RNA (sgRNA) gene was placed under the control of constitutive promoter J23119 (which contains a SpeI restriction site; as on pgRNA [39]) and was edited to encode a SacI restriction site in its upper stem region, the function of which is generally resilient to mutations [40]. These two restriction sites allow simple exchange of the specificity-defining N20 stretch on the sgRNA.
The CRISPR-Cas9 gene cassette was commercially synthesized (Thermo Scientific) and delivered on vector pMA-RQ_csg. To exchange sgRNA target specificity of pMARQ_csg, DNA oligonucleotides containing a 20 nt specificity region with SpeI-and SacI-compatible overhangs (Table 1; N20_aacC1_top/btm; N20_nt_top/btm) were annealed by mixing 10 µl of each 100 µM oligo with 80 µl of annealing buffer (100 mM potassium acetate, 30 mM HEPES, pH=7.5) and heating to 95 °C followed by slow overnight cooling to room temperature. Subsequently, the annealed oligos were phosphorylated using T4 polynucleotide kinase (NEB) according to the manufacturer's instructions. The annealed and phosphorylated oligos were inserted between pMARQ_csg's SpeI and SacI restriction sites following standard molecular cloning protocols, resulting in pMARQ_csg[aacC1] and pMARQ_csg[nt] (Fig. 1c).

pKJK5::csg recombineering
The CRISPR-Cas9 cassette was introduced to pKJK5 using homologous recombineering with an altered version of pDOC-K and pACBSCE plasmids [41]. The following steps were carried out in parallel with pMARQ_csg[aacC1] and pMARQ_csg[nt]. To construct pDOC_csg as a template vector containing the CRISPR cassette, the kanamycin resistance gene was removed from pDOC-K using AvrII and NheI restriction sites, gel extraction (Qiagen gel extraction kit) and religation of the 5.9 kb band. Next, the CRISPR-Cas9 cassette was inserted from pMARQ_csg using restriction sites EcoRI and HindIII to create pDOC_csg.
E. coli DH5α+pKJK5 was transformed with pACBSCE and pDOC_csg following standard procedures for electrotransformation of E. coli. Briefly, log-phase E. coli were washed twice with ice-cold 10 % (w/v) glycerol and concentrated ~30 times. Cells were electroporated by applying 1.8kV in 2 mm gap cuvettes.
Cells were cultured in the presence of Tc+Tmp (pKJK5)+Ap (pDOC_csg)+Cm (pACBSCE) to maintain plasmids, and in the presence of Gluc to prevent leaky λ-red expression.
Then 10 µl of an overnight culture of this recombineering-ready strain were used to inoculate 1 ml LB+Tc+Tmp+Cm+Ap+Gluc at 37 °C and grown at 250 r.p.m. for 2 h in triplicate. The cultures were spun and resuspended in 1 ml LB+Tc+Ara and incubated at We analysed read quality using FastQC v0.11.9 and discarded poor-quality reads using Trimmomatic v0. 39. Assembly was carried out using SPAdes v3.15.2 with the settings --plasmid and --careful. QUAST v5.0.2 was used to try multiple settings and select the best assembly. Finally, SPAdes-generated contigs were visualized using Bandage v0.8.1 and aligned to GenBank-deposited pKJK5 (AM261282.1) and E. coli K12 sequences (NZ_CP010444.1). Contigs that aligned to E. coli K12 were discarded, which yielded a single circular contig encoding pKJK5 and the CRISPR-Cas9 cassette. This contig contained a 127 bp duplication as an artefact of circularization, which we removed as a post-processing step. pKJK5::csg[aacC1] is identical to its theoretical sequence, except for a single-nucleotide deletion in the pKJK5 backbone 12 nt upstream of trfA. pKJK5::csg[aacC1] is deposited on GenBank under the accession number OP921802.

Blocking plasmid uptake in E. coli
Electrocompetent E. coli strains were prepared using standard protocols as described above.
E. coli DH5α+pKJK5::csg[aacC1]/[nt] were electroporated with 500 ng of plasmid DNA (pHERD30T/pHERD20T) in six replicates. Fifty microlitres of transformed cells were plated onto selective plates (LB+Gm/Amp) and transformation efficiency was calculated for each strain (c.f.u. ml −1 µg −1 DNA). Where no colonies could be recovered, transformation efficiency was set to ½ of the limit of detection (transformation efficiency if a single colony was recovered). Plating transformation mixes onto double selective plates (LB+Tc+ Gm/Amp) yielded a similar amount of colonies (not shown).

E. coli conjugation experiments
For liquid mating, single colonies of donors (E. coli DH5α+pKJK5::csg[aacC1]/[nt]) and recipients (E. coli K12::mCherry+pHERD30T) were suspended and grown overnight in 5 ml each LB+Tc or LB+Gm, respectively. Cultures were washed twice with 0.9 % (w/v) NaCl and 50 µl of donors and recipients were co-incubated in fresh 5 ml LB microcosms in six replicates and incubated overnight at 37 °C, 50 r.p.m. The next day, all cultures were frozen in 20 % (w/v) glycerol at −70 °C and plated onto various selective media: LB without selection allows donors and recipients to grow, LB+Km selected for all recipients, LB+Km+Tc selected for recipients that had taken up pKJK5::csg (transconjugants), LB+Km+Gm selected for recipients with target plasmid pHERD30T, and LB+Km+Gm+Tc selected for recipients containing both plasmids. All Table 2. Sequence sources of CRISPR-Cas9 cassette coding and non-coding elements

Cas9
Addgene plasmid #39 312 [46]. Coding sequence only sgRNA Addgene plasmid #44 251 [39]; N20 replaced to target aacC1. Constitutive promoter and terminator as in source. Upper stem edited as described below GFPmut3b [47] Multiple cloning site pBAM1 [48]. The final version is heavily edited to exclude restriction sites used elsewhere Cas9 promoter/terminator Constitutive promoter as found on pBAM1 [48]: bla Ampicillin resistance upstream region (70 nts) with two final nucleotides changed to CC (to create NcoI restriction site for promoter exchange), downstream region (54 nts) as terminator GFP promoter P A1/04/03 as found on GenBank acc. no. DQ493878. Constitutive, LacI-repressible promoter with strong ribosome binding site GFP terminator neo Kanamycin resistance downstream region (29 nts) as found on pBAM1 [48] Homology arms Upper homology: nt 450-550; lower homology: nt 551-651 of dfrA on pKJK5 (GenBank accession AM261282.1) selective plates were also analysed for GFP expression as above, and GFP expression was found to be as expected (GFP+ colonies when pKJK5::csg was selected for). Additional controls (not shown) included donor-only and recipient-only controls, and yielded colonies as expected. Enumerating colonies on selective plates allowed us to calculate the proportions of recipients carrying various plasmids.

Broad host-range barrier to plasmid uptake
Further information on isolates used in this experiment is given in Table S1.
Using E. coli DH5α or E. coli MFDpir as a donor, pKJK5::csg[aacC1]/[nt] transconjugants of bhiF2, C743E1, TV1-2, 6TB-1, Pseudomonas aeruginosa PA14, and of Pseudomonas fluorescens SBW25 were generated and pKJK5::csg[nt]/[aacC1] was selected for and maintained with Tc (except for SBW25, where selection at this Tc concentration failed and transconjugants were selected by identifying GFP+colonies). Next, each strain was made electrocompetent and transformed with 600 ng pHERD30T in six replicates following the E. coli electroporation protocols described above (bhiF2, C743E1, TV1-2, 6TB-1). P. aeruginosa PA14 was made electrocompetent by washing 1 ml aliquots of an overnight culture twice with a 300 mM sucrose solution followed by resuspension in 100 µl 300 mM sucrose. To prepare electrocompetent P. fluorescens cells, the protocol for PA14 was followed with the exception that SBW25 was grown at 28 °C, cultures were grown until log phase in the absence of Tc selection (estimated OD 600 : 0.5-0.6) and then the protocol was started. Pseudomonas cultures were electroporated at 2.5kV in 2 mm gap cuvettes in six replicates, and recovered in 1 ml SOB at 37/28 °C, 250 r.p.m. for 1 h. Samples that arced during electroporation were discarded, yielding n=3-6 for all samples.
Eight hundred microlitres of all strains were plated onto LB+Gm and transformation efficiency calculated as described above. For P. fluorescens SBW25 transformations, only 50 µl of transformed cells were plated, resulting in a higher limit of detection.
As a control for competence, one replicate of each competent strain carrying pKJK5::csg[aacC1] was transformed with pHERD30T_mut, a pHERD30T-derivative that encodes a mutated aacC1 gene and is therefore not targetable by pKJK5::csg. All controls yielded colonies, indicating successful transformation and competence of strains.

Conjugative removal from diverse isolates
In order to assess whether pKJK5::csg could be used to conjugatively remove pHERD30T from this library of isolates, we transformed each of the isolates with pHERD30T after preparing electrocompetent cells as described above.
In parallel, we carried out liquid mating as described above for E. coli and solid-surface filter mating, in a donor : recipient ratio of 100 : 1. In brief, 1 ml each of OD-adjusted donor cultures and of 1 : 100 diluted recipient cultures were concentrated onto a 0.2 µM pore size cyclopore membrane by applying a vacuum, and membranes were placed onto 10 % LB plates and incubated at 37 °C (28 °C for SBW25) overnight (n=5). Membranes were placed into 3 ml 0.9 % NaCl and vortexed to recover cells. In addition to the usual donor-only and recipient-only controls, sterility controls were carried out to check for sterility of the vacuum pump and filter system, and no colonies were recovered on any selective medium.
Cell suspensions of recovered cells after filter mating as well as after liquid mating were plated onto various selective media in 5 µl droplets from a dilution series ranging from 10 0 (undiluted) to 10 −7 . Cultures were plated onto LB (all donors and recipients), LB+Ap (all recipients), LB+Ap+Tc (transconjugants), LB+Ap+ Gm (recipients with target plasmid) and LB+Ap+Tc+Gm (recipients with both plasmids). For PA14 samples, Ap was replaced with Km. For SBW25 samples, Tc was replaced with 120 µg ml −1 tetracycline. PA14 recipient-only controls showed a low amount of small colonies on plates containing Tc, so these plates were not used in any analyses for treatments containing PA14.

Statistical analyses
Data processing, data visualization and statistical analyses were carried out using R software version 4. Generalized linear models (GLMs) were fitted to data as listed below using the base R 'glm' function; additional details are given below and in Table S3. For all models, homogeneous variance of residuals, normal distribution of residuals, explanatory variable collinearity and absence of bias by influential observations were tested by plotting data and using the plot(model) function and these assumptions were found to be upheld. Other model types, link functions and model structures were tested and the models that satisfied assumptions the best during model validation were chosen. Inclusion of other explanatory variables was tested, and non-significant variables were dropped. Where mentioned, a Tukey's post-hoc test was carried out to assess statistical difference between treatment categories. pKJK5::csg conjugative delivery: target plasmid retention (Fig. 3a) First, numerical proportions of >1 were set to 1 to allow for proportional data analysis (one datapoint for the pKJK5::csg[aacC1] treatment when selecting for pKJK5 only). Next, we fitted a binomial GLM with logit link function describing proportion as a function of pKJK5::csg target, selective medium and their interaction. Raw colony counts on plates selecting for recipients (K) were used as weights. The binomial model structure was chosen to suit the proportional data. An inverse Gaussian GLM was fitted with log link function describing target plasmid proportion as a function of isolate identity, pKJK5::csg target and their interaction. Data from filter mating and from liquid mating were modelled separately. The inverse Gaussian model structure was chosen to suit the zero-bounded data; binomial modelling was unavailable for this full dataset due to high variation and proportions exceeding 1. Model coefficients showed significant effects in C743E1 during filter (P=0.002) and liquid (P=1.5×10 −8 ) mating, and for TV1-2 during filter mating (P=0.006). TV1-2 was chosen for further analysis due to likely biological relevance (see below); C743E1's effects are likely attributed to low pHERD30T maintenance regardless of pKJK5::csg treatment.
pKJK5::csg conjugative delivery to TV1-2 by filter mating (Fig. S2) We fitted a binomial GLM with logit link function describing proportion as a function of pKJK5::csg target, selective medium and their interaction. A single high-influence data point was removed for analyses (100 % pKJK5 conjugation efficiency in a single targeting replicate). Raw colony counts on plates selecting for recipients (A) were used as weights. The binomial model structure was chosen to suit the proportional data. All data visualizations and analyses were carried out after setting the proportions below to ½ of the limit of detection (the proportion of transconjugants carrying pHERD30T if a single colony was found on the appropriate selective plates). Analyses of PA14 and of SBW25 samples were excluded due to inaccurate assessment of transconjugants (PA14 recipient-only controls grew in the presence of Tc) and a lack of conjugation, respectively.
We fitted a Gaussian GLM with identity link function describing log-transformed proportion as a function of pKJK5::csg target, recipient identity and their interaction. Data from filter and from liquid mating were modelled separately.